α-Amanitin induced apoptosis in primary cultured dog hepatocytes

Abstract

Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. Awide variety of amatoxins have been isolated; however, α-amanitin (α-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that α-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in α-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with α-AMA at a final concentration of 1, 5, 10 and 20 μM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to α-AMA. There was a clear correlation between hepatocyte viability, concentration of α-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to α-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning. © Polish Histochemical et Cytochemical Society.